Development of a high-titer retrovirus producer cell line capable of gene transfer into rhesus monkey hematopoietic stem cells.

Abstract

Retroviral-mediated gene transfer into primitive hematopoietic cells has been difficult to achieve in largeanimal models. We have developed an amphotropic producer clone that generates' >1010 recombinant retroviral particles (colony-forming units) per ml of culture medium. Autologous rhesus monkey bone-marrow cells were cocultured with either high (2 X 1010 colony-forming units/ml) or low (5 X 106 colony-forming units/ml) titer producer clones for 4-6 days and reinfused into sublethally irradiated animals. The proviral genome was detected in blood and bone-marrow cells from all three animals reconstituted with cells cocultured with the high-titer producer cells. In contrast, three animals reconstituted with bone marrow cocultured with the low-titer producer clone exhibited no evidence of gene transfer.Retroviral-mediated gene transfer (1,2) has been successfully applied to the insertion and expression of various genes in murine stem cells, including the bacterial neomycin resistance (neo) (3-5), human dihydrofolate reductase (6), murine interleukin 3 (IL-3) (7), murine granulocyte-macrophage colony stimulating factor (GM-CSF) (8), human adenosine deaminase (ADA) (9-11) and human P-globin genes (12)(13)(14) (17,18).The efficiency of retroviral-mediated gene transfer into murine stem cells is influenced by the titer ofthe recombinant virus (9), preconditioning of the donor marrow with 5-fluorouracil (5-FU) (3,4,6) and the addition of the hematopoietic growth factors IL-3 (3, 4) and interleukin 6 (IL-6) (13). (22) and GP+E86 (23) cells were maintained in Dulbecco's modified Eagle's medium (DMEM; Biofluids) with 10% newborn calf serum (NCS; GIBCO). A pA317 cell line producing 5 x 106 infectious particles per ml of the N2 (24) virus was generated by standard techniques (25). This cell line was cotransfected with the plasmids pXM-gIL-3 (26), pXM-IL-6 (27), and pMohgr, which contain the gibbon IL-3, the human IL-6, and the hygromycin resistance genes, respectively. 3738The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

A chromatin insulator protects retrovirus vectors from chromosomal position effects

“…Retrovirus vector producer lines were generated essentially as described (24) using the amphotropic packaging line PA317 (25) and the ecotropic packaging line GP ϩ E86 (26). Virus titers were determined by serial dilution and transfer of G418 resistance to naive NIH 3T3 cells as described (27). Clones with the highest titers were further analyzed by Southern blot analysis for intact provirus (methods described below), and for the presence of replication-competent virus by a standard marker-rescue assay (24).…”

“…Retrovirus vector producer lines were generated essentially as described (24) using the amphotropic packaging line PA317 (25) and the ecotropic packaging line GP ϩ E86 (26). Virus titers were determined by serial dilution and transfer of G418 resistance to naive NIH 3T3 cells as described (27). Clones This paper was submitted directly (Track II) to the PNAS office.…”

Retroviral transduction of quiescent haematopoietic cells using a packaging cell line expressing the membrane-bound form of stem cell factor

“…In this context, it could also be of use in targeting retrovirus to cells in close proximity to the producers, by localising growth factor spread or by limiting proliferation to specific subsets of cells bearing receptors. Similar approaches to ours, but using soluble cytokines have been used successfully for retroviral gene transfer to bone marrow progenitor cells 28 and to support human haematopoiesis in long-term culture, using mouse stromal layers that were engineered to produce human cytokines. 29 For the purpose, ultimately, of targeting haematopoietic stem cells, our decision to use SCF as the stimulating growth factor was dictated by a number of lines of evidence suggesting that SCF may be effective in increasing the efficiency of retroviral transfer to PHSC.…”

Differences among nonhuman primates in susceptibility to bone marrow progenitor transduction with retrovirus vectors

“…Vector-containing supernatant stocks were prepared by 24 h incubation of semiconfluent cultures in D8 medium, passed through 0.44 m low protein-binding filters (Millex-HV, Millipore, Bedford, MA, USA), and stored in aliquots at −70°C. Virus preparations from both producer clones had titers ranging from 2 to 5 × 10 6 colony-forming units per milliliter based on end-point titration of G418 resistance on NIH3T3 cells as previously described, 50 and were free of replication-competent virus based on a standard marker-rescue assay. 25 …”